CN114657190A

CN114657190A - Msn2p as negative regulatory factor in improving protein expression in host cells

Info

Publication number: CN114657190A
Application number: CN202210356130.3A
Authority: CN
Inventors: 姚冬生; 林香娜; 刘大岭; 谢春芳; 丁伟秋
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2022-04-06
Filing date: 2022-04-06
Publication date: 2022-06-24
Anticipated expiration: 2042-04-06
Also published as: CN114657190B

Abstract

The invention relates to application of a transcription regulatory factor for eukaryotic gene expression, in particular to application of a transcription regulatory factor Msn2p of a constitutive promoter Pgap. The invention discloses an application of Msn2p as a negative regulatory factor in improving protein expression in host cells, wherein an amino acid sequence of Msn2p is coded by Msn2 gene with a nucleotide sequence of SEQ ID NO. 1; the application is to increase the expression of a protein in a host cell by knocking out Msn2 gene. The application of the invention can enhance the transcription regulation of a constitutive promoter Pgap in a pichia pastoris expression system by reducing repression, thereby improving the expression efficiency and the yield of target protein.

Description

Msn2p as negative control factor in improving protein expression in host cells

Technical Field

The invention belongs to the field of molecular biology and bioengineering, relates to application of a transcription regulatory factor for eukaryotic gene expression, and particularly relates to application of a transcription regulatory factor Msn2p of a constitutive promoter Pgap.

Background

The promoter is one of the most important elements for regulating gene expression, P_AOX1The promoter (inducible) and Pgap promoter (constitutive) are the most representative promoters in the expression of Pichia pastoris foreign proteins.

The expression system of methanol-induced pichia pastoris is a commonly used expression system for expressing most heterologous proteins at present, however, not all the heterologous proteins are suitable for methanol-induced expression, and methanol has great potential safety hazard in large-scale production. Compared with methanol induction, methanol induction is not used when a constitutive pichia pastoris expression system expresses heterologous proteins, but most constitutive promoters are relatively weak in strength, and high protein yield cannot be obtained, so that the application of the constitutive pichia pastoris expression system is limited.

Aiming at the defects of methanol induction, the optimization and modification of a pichia pastoris expression system are a research hotspot in recent years. In the research of promoter modification, new promoter libraries are mainly constructed by various methods at present. Among them, there are many studies to modify PAOX1 by deleting or inserting a cis-acting element in PAOX1, point-mutating the 5' UTR or core promoter region, etc., to result in activation of PAOX1, but these modifications do not appear to be good enough to eliminate inhibition caused by alternative carbon sources such as high levels of glucose and glycerol, and are far from the level of industrial application. For the construction of the Pgap library, the only research is that Qin and the like construct the GAP promoter library by random mutation through error-prone PCR, but the method is random and cannot explain the regulation and control of Pgap. With the application of transcriptome data analysis, the promoter development work is greatly improved. Many reports have been reported on related studies on regulation and improvement of expression intensity of PAOX1, and current studies indicate that methanol can regulate PAOX1 at the transcriptional level by regulating the activity or subcellular localization of multiple trans-acting elements (transcription regulatory factors mainly focused on the PAOX1 promoter or carbon source repression related transcription factors, etc.), thereby affecting the expression of genes related to methanol metabolic pathway. Nevertheless, the mechanisms involved in the regulation of PAOX1 are complex, and the derepression of part of the carbon source does not go beyond the traditional methanol induction in protein expression. At present, the research on promoter regulation is mainly focused on PAOX1, but for the research on exploring Pgap transcription regulation, only Ozge Ata and the like construct a high-expression rhGH strain of a promoter variant by specifically deleting or replicating a Transcription Factor Binding Site (TFBS), and the report of enhancing the yield of a Pgap promoter expression target protein by improving the Pgap promoter transcription regulation in the published literature is almost blank.

According to literature reports, transcription factor Msn2p plays a key role in responding to environmental stress by activating transcription of stress response genes in saccharomyces cerevisiae. Msn2p is assumed to be PAS _ chr2-1_0723 (SEQ ID NO: NC _012964.1) in Pichia pastoris, and the transcriptional regulation of Pgap by the PAS _ chr2-1_0723 has not been reported yet.

Disclosure of Invention

The invention mainly aims to solve the problems and defects of heterologous protein expression in host cells, and provides a transcription factor Msn2p for regulating and controlling the expression of heterologous proteins in host cells, which can enhance the transcription control of a constitutive promoter Pgap in a pichia pastoris expression system by reducing repression, thereby improving the expression efficiency and yield of target proteins.

In the first aspect of the invention, Msn2p is provided as a negative regulatory factor in improving the protein expression in host cells, wherein the amino acid sequence of Msn2p is coded by Msn2 gene with the nucleotide sequence of SEQ ID NO. 1; the application is to increase the expression of a protein in a host cell by knocking out Msn2 gene.

According to the use of the invention, the host cell is selected from: pichia pastoris, Saccharomyces cerevisiae, Candida glycerinogenes.

According to the reports of the prior documents, Pichia pastoris, Saccharomyces cerevisiae, Candida glycerinogenes and the like can all receive yeast expression vectors taking Pgap as a promoter to express heterologous proteins, and therefore, the yeast expression vectors can be used as host cells of the invention.

In a second aspect of the invention, a gene expression cassette for regulating expression of a heterologous protein in a host cell is provided, wherein Pgap is used as a promoter and Msn2 gene is knocked out.

In a third aspect of the invention, a vector is provided, which comprises the gene expression cassette of the invention.

In a fourth aspect of the invention, there is provided a host cell comprising the gene expression cassette of the invention, or comprising the vector of the invention.

In a fifth aspect of the invention, there is provided a method of enhancing protein expression in a host cell, comprising: providing a host cell according to the invention, culturing said host cell under conditions suitable for expression of said heterologous protein, and isolating the expressed protein from the culture medium.

The invention discloses through experiments: msn2 gene is knocked out in a heterologous protein synthesis path with Pgap as a promoter, a transcription factor Msn2p is formed after the sequence is translated and participates in the reverse regulation of the Pgap promoter, and the repression expression of a subsequent exogenous gene with the Pgap as the promoter is removed, so that the non-induced mass accumulation of the target protein is realized.

The gene expression cassette which takes Pgap as a promoter and knocks out Msn2 gene can be constructed, and the knockout of Msn2p transcription inhibiting factors can enhance the transcription of a constitutive promoter Pgap promoter of pichia pastoris, so that exogenous genes in the gene expression cassette are efficiently expressed in the pichia pastoris (or other yeasts which can accept Pgap as a promoter), and the expression strength of the exogenous genes can be obviously improved.

Drawings

FIG. 1 is a map of an Msn2 gene knockout expression cassette; a picture shows a kan gene substitution knockout expression cassette; panel b is a full knockout expression cassette.

FIG. 2 shows the growth rate of xylanase xynB expressed by the strain of the invention (Pichia pastoris), wherein the control group is an un-knocked-out strain; clone 1 and clone 2 were Msn2 knock-out strains.

Detailed Description

The technical solution of the present invention will be further described in detail with reference to the following embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods. The molecular cloning techniques employed in the following examples are described in the molecular cloning guidelines, compiled by J.Sammlung et al, or as recommended by the manufacturer.

Herein, Msn2p denotes a protein expressed by Msn2 gene.

Example 1: construction of Kan gene replacement transcription inhibitor Msn2 gene knockout expression cassette

1. Amplification of upstream homology arms

The invention takes the genome sequence of pichia pastoris as reference, adopts software DNAMAN 8 to design and synthesize two oligonucleotide primers, and amplifies the upstream homology arm sequence of Msn2 gene (the sequence is SEQ ID NO:1) by a PCR method.

The two PCR primers were as follows:

1-F:ACTTACGAGCTCGGCTCAGTGGCTTTCCATCTGTT(SEQ ID NO:4)

1-R:CTTACGCGGATCCGCGTCTAGTTAATCGCAAAC(SEQ ID NO:5)

underlined bases are the SacI and BamHI restriction enzyme sites, respectively.

The PCR reaction system is shown in Table 1 below.

Table 1:

components	Volume (μ L)
		2×Q5 Master Mix	12.5
Primer F(10μM)	1.25
		Primer R(10μM)	1.25
Template DNA	1(100ng)
		ddH₂O	9ul
Total volume	25ul

The PCR program was set up as in Table 2 below.

Table 2:

the PCR amplification product was subjected to 1% agarose gel electrophoresis, and gel recovery was carried out using a DNA recovery kit to obtain a fragment of about 1000 bp.

2. Amplification of downstream homology arms

The invention takes the genome sequence of pichia pastoris as reference, adopts software DNAMAN 8 to design and synthesize two oligonucleotide primers, and amplifies the downstream homology arm sequence of Msn2 gene by PCR method.

The two PCR primers were as follows:

2-F:ACTTATTTGCGGCCGCTTTAAGGAATTGGGAAGGTTTTATATT(SEQ ID NO:6)

2-R:ACTTAGTTTCTTCGTCGACTCTTTTATTGAGCCTGTGTC(SEQ ID NO:7)

underlined bases are SalI and NotI restriction enzyme cutting sites respectively.

The PCR reaction system and PCR program set up the amplification method of the upstream homology arm as described above.

3. Amplification of Kan Gene

In the invention, the Kan gene (kanamycin resistance gene which can express G418 resistance in a cell) of a ppic3.5k plasmid (from Invitrogen company) is used as a reference, two oligonucleotide primers are designed and synthesized by adopting software DNAMAN 8, and the Kan gene is amplified by a PCR method.

The two PCR primers were as follows:

3-F:ACTTACGCGGATCCGCGATGAGCCATATTCAAC(SEQ ID NO:8)

3-R:ACTTACCGGAATTCCGGTTAGAAAAACTCATCGAG(SEQ ID NO:9)

underlined bases are the restriction sites for BamHI and EcoRI respectively.

The PCR reaction and PCR program were set as described above.

After the PCR amplification product was subjected to 1% agarose gel electrophoresis, gel recovery was carried out using a DNA recovery kit to obtain a fragment of about 816 bp.

4. Ligation of the ppic3.5k vector to the Kan fragment

The ppic3.5K plasmid and the Kan gene PCR product were digested with restriction enzymes EcoRI and BamHI, respectively, at 37 ℃ for 20min under the conditions shown in Table 3 below.

Table 3:

components	Volume (μ L)
		10 XCutsmart buffer	1
Plasmid	1 (about 200ng)
		EcoRI、BamHI	0.2
ddH₂O	7.6
		Total volume	10

After electrophoresis of the enzyme digestion product on 1% agarose gel, two target fragments were recovered respectively and ligated with T4DNA ligase, the ligation system is shown in Table 4 below.

Table 4:

and (2) connecting for 12h by using ligase at 16 ℃, transforming DH5a competent cells by using a connection product, amplifying, extracting plasmids by using a plasmid extraction kit, carrying out double enzyme digestion by using EcoRI and BamHI, carrying out electrophoresis, and indicating that two bands of 9kb and 816bp exist, wherein the successful connection is indicated, and the DNA sequencing is carried out to determine the gene as Kan.

5. The upstream homology arm fragment is connected with a ppi3.5k-Kan carrier

The upstream homologous arm fragment and the ppi3.5k-Kan vector are obtained by double digestion with restriction enzymes SacI and BamHI respectively and purification and recovery.

The method for connecting the upstream homology arm fragment and the ppi3.5k-Kan vector is the same as the step 4, the electrophoresis result after the double digestion by SacI and BamHI shows that two bands of 9kb and 1kb are provided, the connection is successful, and the upstream homology arm fragment is determined by DNA sequencing (the sequence is SEQ ID NO: 2).

6. The downstream homology arm fragment is connected with a ppic3.5k- (upstream homology arm) -Kan carrier

The downstream homology arm fragment and the ppic3.5k- (upstream homology arm) -Kan vector are obtained by double digestion, purification and recovery of restriction enzymes EcoRI and NotI respectively.

The method for connecting the downstream homology arm fragment and the ppic3.5k- (upstream homology arm) -Kan vector is the same as that in step 4, the electrophoresis result after double digestion by SalI and NotI shows that two bands of 8kb and 1kb are displayed, the connection is successful, and the downstream homology arm fragment is determined by DNA sequencing (the sequence is SEQ ID NO: 3).

Thus, the Kan gene replacement transcription repressing factor Msn2 gene knockout expression cassette is successfully constructed (as shown in a picture of figure 1).

Example 2: construction of complete knockout expression cassette of transcription repressing factor Msn2 gene

The homology arms above and below the Msn2 gene were amplified using primers 1-F, 1-R, and 2-F, 2-R as in example 1, and the upstream and downstream homology arm fragments were ligated to the ppic3.5k vector by the above-described enzymatic ligation to construct a ppic3.5k- (upstream homology arm) - (downstream homology arm) knockout expression cassette.

Example 3: pichia pastoris genome Msn2 knock-out

In order to improve the integration efficiency of the single copy expression cassette on the chromosome of pichia pastoris, the knockout expression cassette is linearized by restriction enzymes SacI and NotI and purified and recovered by a kit. The recipient bacterium of the experiment is pichia pastoris SMD1168 (recombinant bacterium containing xylanase xynB gene inserted after Pgap, EX6), the knockout expression cassette of example 1 is electrically transformed, and then screened by using a G418 plate containing 0.3mg/mL, and genome PCR identification is carried out. Example 2 knock-out expression cassettes were electrotransformed and screened using YPG plates and genomic PCR identification was provided.

The PCR product sequencing result shows that the screened strain is a positive clone which successfully knocks out Msn 2.

Example 4: msn2 knock-out strain constitutive promoter Pgap driven heterologous protein expression assay

The positive clone 1 selected in example 1 and the positive clone 2 selected in example 2 were fermented at 28 ℃ and 200rpm for 72 hours. Meanwhile, pichia pastoris (EX6) containing xynB genes is used as a control, after culture for 72h, supernatant is taken to carry out SDS-PAGE electrophoresis detection, and the expression level of xylanase xynB is analyzed.

The results are shown in fig. 2, the expression level of the positive clone 1 picked after knocking out Msn2p transcription factor is increased by 118% compared with the control group, and the expression level of the positive clone 2 is increased by 50% compared with the control group.

SEQUENCE LISTING

<110> river-south university

Application of <120> Msn2p as negative regulatory factor in improving protein expression in host cells

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<170> PatentIn version 3.5

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Claims

1, Msn2p as negative regulation factor in improving the host cell protein expression application, characterized by, the amino acid sequence of Msn2p is encoded by Msn2 gene whose nucleotide sequence is SEQ ID NO. 1; the application is to increase the expression of a protein in a host cell by knocking out Msn2 gene.

2. The use according to claim 1, wherein said host cell is selected from the group consisting of: pichia pastoris, Saccharomyces cerevisiae, Candida glycerinogenes.

3. A gene expression cassette for regulating expression of a heterologous protein in a host cell, wherein the Msn2 gene is knocked out using Pgap as a promoter.

4. A vector, characterized by: which contains the gene expression cassette of claim 2.

5. A host cell, characterized in that: comprising the gene expression cassette of claim 2 or comprising the vector of claim 3.

6. A method of enhancing protein expression in a host cell, comprising: providing a host cell according to claim 4, culturing said host cell under conditions suitable for expression of said heterologous protein, and isolating the expressed protein from the culture medium.